Project Summary/Abstract Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor despite extensive treatment measures and intense research effort. There is an urgent need for innovative therapies that can improve upon the current ?largely ineffectual? clinical standard of care. Motivated by this need, the objective of this proposal is to synthesize and test a novel, GBM-specific, highly potent immunotoxin and prolong its bioavailability by sustained release from an intratumorally injected, gel-like polypeptide depot. The bioactive component of the drug ?an Affibody-toxin fusion? is internalized upon high affinity binding of the targeting protein ?the Affibody? to GBM cell-specific receptors. The bacterial toxin fused to the Affibody is an exceptionally powerful drug, as it is highly cytotoxic regardless of cell cycle status. However, injection of biologics is limited by their high solubility and rapid clearance from the tumor. To address this problem, we will create a depot the enables sustained intratumoral release by genetic fusion of a third component ?an elastin-like polypeptide (ELP)? a thermally responsive peptide polymer that transitions from a soluble to an insoluble coacervate phase upon in vivo injection, leading to the formation of an intratumoral drug depot. The kinetics of release from this depot will be optimized using a library of ELPs with a range of phase transition behaviors. In preliminary studies, we have demonstrated picomolar potency of our Affibody-immunotoxin-ELP fusion in GBM cell lines in vitro and anticipate that the intratumoral injection of this immunotoxin depot formulation in vivo will result in slow dissolution into the surrounding tissue and enhanced efficacy compared to existing immunotoxin treatments that can only achieve sustained application through delivery by external osmotic pumps. The proposed work is significant because it improves upon previously investigated GBM-specific immunotoxins by: 1) enhancing targeting by use of a structurally robust Affibody, 2) increasing valency and therefore avidity through recombinant fusion of multiple Affibody domains, 3) improving biocompatibility with an engineered, non- immunogenic toxin; and 4) creating a fusion that can be injected into tumor tissue as a liquid but which instantaneously transitions into an insoluble and viscous material driven solely by body heat. The proposal is innovative in its design where the targeting moiety, drug and depot are modular ?enabling easy swapping of different modules? and can be engineered into a single polypeptide that can be recombinantly produced at high yield in bacteria and are easily purified, thereby making this a highly generalizable and clinically translational technology.